U.S. patent number 4,735,902 [Application Number 06/788,830] was granted by the patent office on 1988-04-05 for stabilized composition containing inositoltriphosphate.
Invention is credited to Matti Siren.
United States Patent |
4,735,902 |
Siren |
April 5, 1988 |
Stabilized composition containing inositoltriphosphate
Abstract
A stabilized composition comprising an organic substrate, such
as a pharmaceutical or biological system subject to degradation by
oxidation and/or free radicals and a method for production
thereof.
Inventors: |
Siren; Matti (CH-6596 Gordola,
CH) |
Family
ID: |
27355315 |
Appl.
No.: |
06/788,830 |
Filed: |
October 18, 1985 |
Foreign Application Priority Data
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Oct 23, 1984 [SE] |
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8405295 |
Jun 26, 1985 [SE] |
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8503164 |
Jun 26, 1985 [SE] |
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8503165 |
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Current U.S.
Class: |
435/188;
424/278.1; 426/52; 426/654; 514/23; 514/44R; 514/5.9; 514/54;
514/573; 987/229 |
Current CPC
Class: |
A23B
7/154 (20130101); A23L 3/3553 (20130101); C07F
9/117 (20130101); A61K 47/24 (20130101); A61K
31/66 (20130101) |
Current International
Class: |
A23B
7/154 (20060101); A23B 7/14 (20060101); A23L
3/3553 (20060101); A23L 3/3463 (20060101); A61K
31/66 (20060101); A61K 47/24 (20060101); C07F
9/00 (20060101); C07F 9/117 (20060101); C12N
009/96 () |
Field of
Search: |
;435/188,183 ;252/400.2
;514/970,3,23,44,573 ;426/654,52 ;424/85,88 |
References Cited
[Referenced By]
U.S. Patent Documents
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2723938 |
November 1955 |
Buckwalter et al. |
3591665 |
July 1971 |
Kimura et al. |
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Other References
Tomlinson et al., Biochemistry, vol. 1, No. 1, pp. 166-171, (Jan.
1962). .
Kerr et al., Archives of Biochemistry and Biophysics, vol. 96, pp.
347-353, (1962)..
|
Primary Examiner: Shapiro; Lionel M.
Attorney, Agent or Firm: Scully, Scott, Murphy &
Presser
Claims
I claim:
1. A stabilized composition comprising an organic substrate subject
to degradation by oxidation and/or free radicals and between about
0.01% to about 2% by weight, based on the total weight of the
composition, of inositol triphosphate (IP.sub.3).
2. A composition according to claim 1, wherein the organic
substrate is a pharmaceutical or biological system.
3. A composition according to claim 2, wherein the pharmaceutical
is selected from the group consisting of insulin, vaccine,
hyaluronic acid, intralipid and prostaglandin.
4. A composition according to claim 2, wherein the biological
system is selected from the group consisting of nucleic acids,
carbohydrates, lipids and proteins.
5. A composition according to claim 1 containing additionally a
minor amount of inositoltetraphosphate (IP.sub.4) and
inositoldiphosphate (IP.sub.2).
6. A composition according to claim 1, wherein the IP.sub.3
contains at least one of D-myo-inositol-1,2,6-triphosphate,
D-myo-inositol-1,2,5-triphosphate, myo-inositol-1,2,3-triphosphate,
D-myo-inositol-1,4,5-triphosphate and
L-myo-inositol-1,3,4-triphosphate.
7. A composition according to claim 6, wherein the IP.sub.3 is a
salt, the cation of which is selected from the group consisting of
alkali metals and alkaline earth metals.
8. A method of stabilizing an organic substrate which comprises
adding between about 0.01% to about 2% by weight of inositol
triphosphate (IP.sub.3) to an organic substrate subject to
oxidation and/or free radical degradation.
9. A method according to claim 8, wherein the organic substrate is
a pharmaceutical or biological system.
10. A method according to claim 8, wherein the pharmaceutical is
selected from the group consisting of insulin, vaccine, hyaluronic
acid, intralipid and prostaglandin.
11. A method according to claim 8, wherein the biological system is
selected from the group consisting of DNA, RNA, nucleic acids,
biological tissue, carbohydrates, lipids, protein such as enzyme,
micro-organisms, seeds, plant parts, spores, fruits and food
stuffs.
12. A method according to claim 8, wherein additionally a minor
amount of inositoltetraphosphate (IP.sub.4) and inositoldiphosphate
(IP.sub.2) is added.
13. A method according to claim 8, wherein the IP.sub.3 contains at
least one of D-myo-inositol-1,2,6-triphosphate,
D-myo-inositol-1,2,5-triphosphate, myoinositol-1,2,3-triphosphate,
D-myo-inositol-1,4,5-triphosphate and
L-myo-inositol-1,3,4-triphosphate.
14. A method according to claim 8, wherein the IP.sub.3 is a salt,
the cation of which is selected from the group consisting of alkali
metals and alkaline earth metals.
Description
The present invention relates to a stabilized composition
comprising an organic substrate, such as a pharmaceutical or
biological system subject to degradation by oxidation and/or free
radicals, and a method for production thereof.
It is well-known that biological materials and pharmaceuticals
often have a rather limited stability even if they are stored in a
dark and cold space. Among the pharmaceuticals having such a low
stability, insulin, vaccine, hyaluronic acid, intralipid and
prostaglandin can be mentioned. In addition, many other organic
substrates susceptible to oxidative or free radical degradation are
well-known to those skilled in the art and include e.g. fats, oils,
ethylenically unsaturated compounds, derivatives and polymers e.g.
acrylate compounds and resins, polyvinylacetate,
polyvinylpyrrolidone, vinylacetate, vinylpyrrolidone and the
like.
It is well-known that the formation of free radicals during storing
and/or use of many pharmaceuticals and biological materials causes
a degradation of these products. The free radicals namely cause an
oxidation, which in its turn results in a degradation of the
pharmaceutical or the biological material. Similar mechanisms are
responsible for the lack of stability of other organic substrates,
such as those mentioned above.
Accordingly, the presence of free radicals can explain why
hyaluronic acid for example is effective at application of eyes but
not at injection in joints for treatment of rheumatism. Free
radicals are formed in the eyes to a slight extent. However, on the
other hand, in the joints, free radicals are formed in a
considerable amount. Therefore, the hyaluronic acid is broken down
or degraded by free radicals in the joint fluids before it has
given the desired effect.
In addition to the negative effect on the stability of
pharmaceuticals and biological materials, free radicals can also
increase the toxicity of said products, which of course is a very
serious problem.
A very intensive research effort has been carried out for many
years to find an effective and non-toxic stabilizer for organic
substrates, such as pharmaceuticals and biological materials. In
general, said work has not given the desired result.
In the U.S. Pat. No. 2,723,938 another kind of stabilization than
that according to the present invention is disclosed. Thus,
according to said patent the use of inositolhexaphosphates
(IP.sub.6), especially sodium phytate for stabilizing
dispersability of aqueous suspensions of insoluble penicillin even
after prolonged storage is shown. The use of said sodium phytate is
said to insure that brief manual shaking will restore a state of
complete and uniform dispersion of the penicillin. However, no
effectiveness in stabilization against oxidative degradation caused
by free radicals is reported. When compared with IP.sub.3
stabilizers of this invention, IP.sub.6 is found to be less
effective as a stabilizer against degradation of organic
substrates. In addition, the use of IP.sub.6 is also seriously
limited because of the impact on the mineral balance in animals
including humans, thus limiting use thereof in compositions to be
administrated to human hosts.
According to present invention a stabilized composition comprising
organic substrates such as pharmaceuticals and biological systems
has been provided. The stabilized composition is characterized in
that it contains inositoltriphosphate, IP.sub.3, in a stabilizing
amount.
The appropriate stabilizing amount of IP.sub.3 should be determined
by routine experimentation to obtain optimum values. For example
accelerated aging test can be performed with a test substrate at
various levels of IP.sub.3 and the optimum level be determined
thereby. In general, at least 0.001% by weight of IP.sub.3 based on
the weight of the composition will provide some beneficial effect.
Usually from 0.01-2% by weight will be employed.
The stabilizer is mainly intended to be used against degradation
caused by free radicals. Such free radicals can be formed in
different ways, for instance by metals, such as iron, aluminium and
cadmium, and by radiation.
However, the stabilizer is intended also to be used against
degradation caused by oxidation and hydrolysis. The oxidation can
be caused by free radicals as mentioned above. However, oxidation
can depend on other mechanisms too. Therefore, the invention covers
stabilization against oxidation, hydrolysis or radiation, whatever
mechanism lies behind said reaction.
The stabilizer can be used for stabilizing many different
pharmaceuticals, of which insulin, vaccines, hyaluronic acid,
intralipid, prostaglandin and hormones can be mentioned.
Also a lot of different biological materials can be stabilized
according to the invention. However, preferably the biological
material is selected from DNA, recombinant DNA, RNA, nucleic acids,
biological tissue, transplants, carbohydrates, lipids, membranes,
proteins, such as enzymes and plasma proteins, culture media for
micro-organisms, cell culture media, blood containing substrates,
blood for transfusion, nutrient substrates, insemination media,
micro-organisms, seeds, plant parts, spores, fruits and food
stuffs.
According to one suitable method for the production of IP.sub.3 a
material containing IP.sub.6 is broken down enzymatically with
phytase enzyme. The IP.sub.6 can be provided either as pure
material or in the form of an IP.sub.6 containing source, such as
wheat bran. Phytase enzyme can be found for instance in plants,
seeds and micro-organisms.
By the enzymatic treatment of the IP.sub.6 a hydrolysis takes place
resulting in a mixture of different lower inositolphosphates, i.e.
inositolpentaphosphate (IP.sub.5), inositoltetraphosphate
(IP.sub.4), inositoltriphosphate (IP.sub.3), inositoldiphosphate
(IP.sub.2) and inositolmonophosphate (IP.sub.1).
Usually, the hydrolysis is carried out at a temperature of
20.degree.-70.degree. C. and a pH of 4 to 8. The hydrolysis is
suitably stopped when the liberation of about 30-60% of the total
ester phosphorus has been achieved. At said stage a high proportion
of the desired IP.sub.3 isomer or isomers has been formed by
hydrolysis of the IP.sub.6 containing material.
The mixture of inositolphosphates obtained may hereafter be
separated by chromatography to isolate the IP.sub.3 -containing
fraction. Preferably, this is made in a column. If the IP.sub.3
fraction contains more than one isomer, these isomers are separated
in another subsequent chromatographic separation step.
The IP.sub.3 can be obtained as a salt or as an acid thereof. The
salt form is preferred, since it is easier to produce in pure and
concentrated form than the acid.
The salt form of the IP.sub.3 isomer is readily obtainable from the
acid form using standard procedures. Thus, there can be prepared
salts, such as alkali metal and alkaline earth metal salts, e.g.
lithium, sodium, potassium, calcium or magnesium. However, also the
aluminium, zinc and iron salts are very useful as well as the
NH.sub.4.sup.+ and organic amine salts. Exemplary amines are
triethanolamine, diethanolamine, triisopropanolamine,
N,N-dimethyl-2-amino-2-methyl-1-propanol, N,N-dimethylethanolamine,
tetrabutylamine and cyclohexylamine. Also other salts might be
used. Especially preferred salts are those which are
physiologically acceptable.
The invention is not restricted to any particular isomer of
IP.sub.3. Consequently, all individual isomers of IP.sub.3 and
mixtures thereof are included in the above definition, IP.sub.3.
However, preferably the stabilized composition comprises at least
one of D-myo-inositol-1.2.6-triphosphate,
D-myo-inositol-1.2.5-triphosphate, myo-inositol-1.2.3-triphosphate,
D-myo-inositol-1.4.5-triphosphate and
L-myo-inositol-1.3.4-triphosphate. Of these isomers
D-myo-inositol-1.2.6-triphosphate is preferred.
When using yeast, preferably baker's yeast as a phytase source,
only one isomer of IP.sub.3 is obtained; namely
D-myo-inositol-1.2.6-triphosphate. Especially when the composition
comprises pharmaceuticals it is generally preferred to use the
isomer or isomers of IP.sub.3 in substantially pure form. The
stabilizer component of the composition can consist wholly or
essentially of IP.sub.3.
The stabilizer is non-toxic and very efficient.
Sometimes the composition can also contain a minor amount of other
inositolphosphates, especially inositoldiphosphate, IP.sub.2 and
inositoltetraphosphate, IP.sub.4 in addition to IP.sub.3. This is
particularly the case where seeds, plant parts, spores, fruits and
foodstuff are to be stabilized according to the invention. IP.sub.2
and IP.sub.4 can be presented in acid as well as in salt form.
The IP.sub.3 -isomers mentioned above have the following
formulas:
D-myo-inositol-1.2.6-triphosphate of the formula ##STR1## where X
is hydrogen, at least one univalent, divalent or multivalent
cation, or a mixture thereof, n is the number of ions, and z is the
charge of the respectively ion;
D-myo-inositol-1.2.5-triphosphate of the formula ##STR2## where X,
n and z have the above mentioned meaning;
myo-inositol-1.2.3-triphosphate of the formula ##STR3## where X, n
and z have the above mentioned meaning;
L-myo-inositol-1.3.4-triphosphate of the formula ##STR4## where X,
n and z have the above mentioned meaning; and
D-myo-inositol-1.4.5-triphosphate of the formula ##STR5## where X,
n and z have the above meaning.
In each of the above formulas n ranges between 6 to 1 inclusive and
z ranges from 1 to 6 inclusive. Preferably, n is between 3 to 6
inclusive and z is 3, 2 or 1.
The invention will be explained further in detail in connection
with the embodiment examples below, of which examples 1-5 show that
IP.sub.3 prevents or reduces the formation of free radicals.
Example 6 illustrates preservation of some fruits and vegetables at
the addition of IP.sub.3. Example 7 shows that an addition of
IP.sub.3 to an enzyme gives a remarkable retention of the enzyme
activity at storage. Examples 8-14 show production of IP.sub.3 and
the separation thereof into different isomers.
EXAMPLE 1
An aqueous solution containing 0.3 mM FeCl.sub.3, 5.0 mM
ethylenediaminotetraacetic acid (EDTA), 50 mM tris
(hydroxymethyl)-aminomethan (TRIS) and 1.0 M NaN.sub.3 was
prepared. In the solution the complex Fe.sup.3+ --EDTA--N.sub.3 was
formed.
A maximum in absorption of light was detected at the wavelength of
409 nm.
Another aqueous solution containing 0.3 mM FeCl.sub.3, 50 mM EDTA
and 50 mM TRIS was prepared. In the solution the complex Fe.sup.3+
--EDTA--H.sub.2 O was formed. There was no maximum in absorption of
light detected at the wavelength of 409 nm.
The above difference in result depends on that N.sub.3.sup.-
competitively replaces one water molecule which binds to the
Fe.sup.3+ --EDTA--complex. This in turn shows that the Fe.sup.3+
--EDTA--complex has a binding site, which is occupied by a
dissociable water-molecule.
It is further known that iron catalyses the formation of
hydroxylradicals. For the formation of these the binding of one
water molecule to iron is required.
This means that EDTA in the EDTA--Fe.sup.3.sup.+ --complex can not
inhibit the formation of hydroxyl radicals catalysed by iron.
The above experiment was repeated with the difference that the EDTA
was substituted with IP.sub.3.
No maximum in absorption was obtained at the wavelength 409 nm.
This result means that the Fe.sup.3+ --complex with IP.sub.3 does
not bind water. Therefore the formation of free radicals is
prevented.
EXAMPLE 2
A reaction mixture consisting of 48 mmol KH.sub.2 PO.sub.4, 2 mmol
Na-ascorbate, 0.1 mmol H.sub.2 O.sub.2, 0.5 mmol Fe and 1.7 mmol
deoxyribose was incubated at 37.degree. C. for 1 hour. Similar
reactions mixtures including EDTA 1 mmol or inositol-tri-phosphate
(IP.sub.3) 1 mmol were similarly incubated. The IP.sub.3 used was
D-myo-inositol-1.2.6-triphosphate.
After incubation 1.65 ml thiobarbituric acid in 50 mmol NaOH and
1.65 ml 2.8% trichloroacetic acid was added to 2 ml of the reaction
mixture. The mixture was heated to 100.degree. C. for 20 minutes
and the absorbance at B 532 nm was measured with water as a
blank.
The experiments were performed with iron in the form of Fe.sup.2+
(Fe(NH.sub.4)SO.sub.4) and Fe.sup.3+ (FeCl.sub.3). The results were
as follows:
Production of free radicals catalyzed by Fe.sup.2+ and Fe.sup.3+ in
the presence of IP.sub.3 or EDTA, expressed as absorbance at 532
nm.
______________________________________ Group Fe.sup.2+ Fe.sup.3+
______________________________________ Control 0.76 0.79 EDTA 2.2
1.86 IP.sub.3 0.46 0.43 ______________________________________
These results show that the formation of free radicals in the
reaction mixture was diminished by 40% after addition of IP.sub.3.
The addition of EDTA had an opposite effect. It strongly increased
production of free radicals. Thus IP.sub.3 was shown to reduce
iron-dependent formation of free radicals. EXAMPLE 3
Lipid peroxidation was studied in lipid micelles. The following
reaction mixture was incubated for 2 hours at 37.degree. C.:
0.4 ml Clark-Lubs buffer pH 5.5
0.2 ml phospholipid liposomes
0.1 ml IP.sub.3 0.5-5 mM or 0.1 ml H.sub.2 O
0.1 ml Fe.sup.2+ 1 mM or 0.1 ml H.sub.2 O
0.1 ml Al.sup.3+ 4 mM or 0.1 ml H.sub.2 O
0.1 ml H.sub.2 O
The IP.sub.3 was D-myo-inositol-1.2.6-triphosphate. After
incubation, 0.5 ml of thiobarbituric acid +0.5 ml 25% HCl was added
and the mixture was heated at 100.degree. C. for 15 minutes. 1 ml
lubrol PX 1% (Sigma) was added and lipid peroxidation was measured
by measuring absorbance at 532 nm. The results were as follows:
______________________________________ Concentration, mM Absorbance
Experiment Fe.sup.2+ Al.sup.3+ IP.sub.3 532 nm
______________________________________ 1 0.1 0 0 0.36 2 0 0.4 0
0.12 3 0.1 0.4 0 0.89 4 0.1 0.4 0.5 0.36 5 0.1 0 0.5 0.30 6 0.1 0
0.4 0.26 7 0.1 0 0.2 0.29 8 0.1 0 0.1 0.28 9 0.1 0 0.05 0.27 10 0 0
0 0.13 ______________________________________
Fe.sup.2+ caused lipid peroxidation (group 1 vs 10). Al.sup.3+
itself caused no peroxidation (2 vs 10) whereas the combination of
Fe.sup.2+ +Al.sup.3+ caused much stronger peroxidation than
Fe.sup.2+ alone (1 vs 3). Addition of IP.sub.3 completely prevented
the interaction between Fe.sup.2+ and Al.sup.3+ (3 vs 4). In
systems with only Fe.sup.2+, IP.sub.3 caused marked reduction in
radical formation (1 vs 5-9).
EXAMPLE 4
Lipid peroxidation was studied in lipid micelles. The following
reaction mixture was incubated for 2 hours at 37.degree. C.:
0.4 ml Clark-Lubs buffer pH 5.5
0.2 ml phospholipid liposomes
0.1 ml IP.sub.3 10 mM or 0.1 ml H.sub.2 O
0.1 ml Fe.sup.2+ 1 mM
0.1 ml Cd.sup.2+ 1 mM or 1 ml Pb.sup.2+ 1 mM or 0.1 ml H.sub.2
O
0.1 ml H.sub.2 O
The IP.sub.3 was D-myo-inositol-1.2.6-triphosphate. After
incubation, 0.5 ml of thiobarbituric acid +0.5 ml 25% HCl was added
and the mixture was heated at 100.degree. C. for 15 minutes. 1 ml
Lubrol PX 1% (Sigma) was added and lipid peroxidation was measured
by measuring absorbance at 532 nm. The results were as follows:
______________________________________ Concentration, mM Absorbance
Experiment Cd.sup.2+ Pb.sup.2+ IP.sub.3 532 nm
______________________________________ 1 0 0 0 0.63 2 0.1 0 0 1.08
3 0.1 0 1.0 0.73 4 0 0.1 0 1.79 5 0 0.1 1.0 1.32
______________________________________
The lipid peroxidation caused by Fe.sup.2+ (group 1) was strongly
increased by Cd (2) and by Pb (4). The effects of both these metals
was strongly counteracted by IP.sub.3 (3 vs 2 and 5 vs 4).
EXAMPLE 5
Reaction mixtures with the following compositions were incubated
for 5 minutes at 37.degree. C.:
______________________________________ KH.sub.2 PO.sub.4 buffer pH
7.4 20 mM EDTA 0.1 mM Salicylate 1 mM Ascorbate 1 mM H.sub.2
O.sub.2 3.3 mM Fe.sup.3+ 0.05 mM IP.sub.3 0, 2.5, 5 or 10 mM
______________________________________
The products formed by oxidation of salicylate were quantified with
HPLC. The IP.sub.3 was D-myo-inositol-1.2.6-triphosphate.
The system studies radical scavenging. Under these reaction
conditions, all Fe.sup.3+ will form complex with EDTA. The Fe--EDTA
complex will induce free radical formation, and the ability of
IP.sup.3 to prevent oxidation of salicylate is studied.
The results of the experiment were:
______________________________________ Concentration Relative
amount of of IP.sub.3, mM salicylate oxidized
______________________________________ 0 100 2.5 44 5 43 10 19
______________________________________
Thus, IP.sub.3 is able to act as a radical scavenger, thereby
preventing free radical induced damage to other molecules.
EXAMPLE 6
Preservation of some fruits and vegetables at the addition of
IP.sub.3.
4 g of fresh potatoes, bananas and apples respectively were sliced
in 10 pieces each. 5 pieces of the same fruit or vegetable were put
into each of 5 different beakers. Three of the beakers were filled
with 15 ml of an aqueous solution of IP.sub.3 and three additional
beakers with 15 ml of an aqueous solution of IP.sub.6, in such a
way that each 5 pieces of fruit and vegetable were exposed either
to pure water or water with IP.sub.3 and IP.sub.6 respectively. The
content of IP.sub.3 and IP.sub.6 respectively in the water was 1.0
g/l.
The samples were allowed to stand in room temperature for 15 hours.
After this period the colour of the samples was inspected and the
following data were found:
______________________________________ Potato Banana Apple
______________________________________ No IP.sub.3 added brown
colour brown colour brown colour IP.sub.3 added very slight very
slight slight brown brown brown IP.sub.6 added brown colour brown
colour brown colour ______________________________________
The results show that IP.sub.3 has a preservative effect on the
fruits and vegetables investigated, whereas IP.sub.6 has no such
effect.
EXAMPLE 7
Enzyme activity at the addition of IP.sub.3.
The activity of aldolase with respectively without addition of
IP.sub.3 was measured as a function of time.
Aldolase degrades fructose-1.6-diphosphate (FDP) to
dihydroxyacetonephosphate. This substrate is further reacted by
.alpha.-glycerophosphatedehydrogenase (GDH) in the presence of
nicotinamide adenine dinucleotide, reduced form (NADH) to
.alpha.-glycerophosphate and nicotinamide adenine dinucleotide,
oxidized form (NAD). By measuring the decline of the UV-absorption
at 340 nm for the reaction NADH to NAD the activity of the enzyme
is determined.
The aldolase was stored at 25.degree. C. and the activity was
measured initially and after 72 hours with and without addition of
IP.sub.3 (4 g/l).
0.5 ul aldolase (A1893 from Sigma Chemical Co, 0.2 U/ml) was mixed
with 2.75 ml of a buffer pH 7.5 consisting of 0.10 g KH.sub.2
PO.sub.4, 0.74 K.sub.2 HPO.sub.4, 196 KCH.sub.3 CH.sub.2 OO, 50 mg
FDP and 8 mg NADH per 100 ml buffer. 1.47 ul GDH was further added
and the total volume was diluted to 3.0 ml. The determination of
the activity was performed at 30.degree. C.
The following results were obtained:
______________________________________ Time for storage at
25.degree. C. No IP.sub.3 added IP.sub.3 added
______________________________________ 0 0.12 0.12 decline of
absorbance/min 72 hours 0.05 0.07 decline of absorbance/min
______________________________________
The results show that the activity of the enzyme was improved by
about 40% after addition of IP.sub.3 when the activity was
determined after 72 hours storage at 25.degree. C.
EXAMPLE 8
Hydrolysis of sodium phytate with wheat phytase and fractionation
of a mixture of inositolphosphates.
A 1.6 gram quantity of sodium phytate (from corn, Sigma Chemical
Co) was dissolved in 650 ml sodium acetate buffer, pH 5.2. 2.7 gram
wheat phytase (EC 3.1.3.26, 0.015 U/mg, from Sigma Chemical Co) was
added and the mixture was incubated at 38.degree. C.
The dephosphorylation was followed by determining the inorganic
phosphorus released. After 3 hours when 50% inorganic phosphorus
was liberated the hydrolysis was stopped by adding 30 ml ammonia to
pH 12. A liquid mixture containing inositolphosphates was
obtained.
350 ml of the mixture was passed through an ion-exchange column
(Dowex 1, chloride form, 25 mm.times.250 mm) and eluted with a
linear gradient of hydrochloric acid (0-0.7 N HCl). Aliquots of
eluted fractions were completely hydrolyzed in order to determine
the contents of phosphorus and inositol. The peaks correspond to
different inositolphosphates i.e. a peak with the ratio of
phosphorus to inositol of three to one consists of
inositoltriphosphate etc. Two fractions with the ratio of
phosphorus to inositol of three to one were obtained.
EXAMPLE 9
Fractionation of inositoltriphosphates.
100 ml of the first fraction obtained in Example 8 with a
phosphorus/inositol ratio of three to one was neutralized and
precipitated as a bariumsalt after addition of 10% excess of 0.1 M
bariumacetate solution. 600 mg of the precipitated salt was
dissolved in 50 ml 0.18 N hydrochloric acid. The solution was
separated on an ion-exchange column (Dowex 1, chloride form, 25
mm.times.2500 mm) with diluted hydrochloric acid as eluent.
Aliquots of eluted fractions were analyzed for phosphorus. Three
peaks consisting of isomers of inositoltriphosphates can be
seen.
EXAMPLE 10
Structural determination of isomers of inositol-triphosphates with
NMR.
The three peaks obtained in Example 9 were analyzed by H-NMR. Data
show that the peaks consist of myo-inositol-1.2.6-triphosphate,
myo-inositol-1.2.3-triphosphate and myo-inositol-1.3.4-triphosphate
respectively.
The second fraction obtained in Example 18 with a
phosphorus/inositol ratio of three to one was analyzed by H-NMR.
Data show that the fraction consists of
myo-inositol-1.2.5-triphosphate.
EXAMPLE 11
Determination of optical isomers of inositol-triphosphates.
20 mg of the compounds determined with NMR according to Example 10
to be myo-inositol-1.2.6-triphosphate and
myo-inositol-1.3.4-triphosphate were further chromatographed on a
chiral column based on acetylated cellulose (20 mm.times.300 mm
from Merck) with a mixture of ethanol and water as eluent. The
fractions were analyzed with a polarimeter. As can be seen each
compound consists of one optical isomer,
D-myo-inositol-1.2.6-triphosphate and
L-myo-inositol-1.3.4-triphosphate respectively.
EXAMPLE 12
Hydrolysis of sodium phytate with baker's yeast and fractionation
of a mixture of inositolphosphates.
A 0.7 gram quantity of sodium phytate (from corn, Sigma Chemical
Co) was dissolved in 600 ml sodium acetate buffer pH 4.6. 50 gram
of baker's yeast from Jastbolaget, Sweden (dry substance: 28%,
nitrogen content: 2%, phosphorus content: 0.4%) was added with
stirring and incubation was continued at 45.degree. C. The
dephosphorylation was followed by determining the inorganic
phosphorus released. After 7 hours when 50% inorganic phosphorus
was liberated the hydrolysis was stopped by adding 30 ml of ammonia
to pH 12. The suspension was centrifuged and the supernatant was
collected.
400 ml of the supernatant was passed through an ion-exchange column
(Dowex 1, chloride form, 25 mm.times.250 mm) and eluted with a
linear gradient of hydrochloric acid (0-0.7 N HCl).
Aliquots of eluted fractions were completely hydrolyzed in order to
determine the contents of phosphorus and inositol. The peaks
correspond to different inositolphosphates i.e. a peak with the
ratio of phosphorus to inositol of three to one consists of
inositoltriphosphates etc.
EXAMPLE 13
Structural determination of isomers of inositoltriphosphate.
The fraction obtained in Example 12 with a phosphorus/inositol
ratio of three to one was neutralized and evaporated before
analysis with H-NMR. Data show that the peak consists of
myo-inositol-1.2.6-triphosphate.
EXAMPLE 14
Determination of optical isomers of myo-inositol-triphosphate.
The same method was used as described in Example 11 with the
difference that 10 mg of the compound determined with NMR according
to Example 13 was analyzed. As can be seen the compound consists of
one optical isomer, D-myo-inositol-1.2.6-triphosphate.
For purposes of further understanding the invention, formulas are
given below of some of the IP.sub.3 -isomers of the invention.
Formulas are also given for IP.sub.6, IP.sub.5, IP.sub.4 and
IP.sub.2.
The lower phosphate-esters of myoinositol are named depending on
where the phosphoric acid groups are situated on the inositol ring,
with the numbering giving as low position numbers as possible. L
and D stand for clockwise and counterclock-wise counting
respectively, and are used depending on which result gives the
lowest position number. The carbon atom which has an axial
phosphoric acid group always has the position number 2. The
structural formulae below are simplified to the acid form.
______________________________________ ##STR6## myo-inositol;
C.sub.6 H.sub.6 (OH).sub.6 ##STR7## ##STR8##
1.2.3.4.5.6-hexakis-(dihydrogen- phosphate)-myo-inositol alterna-
tively myo-inositol hexakis (phosphate) or IP.sub.6 ##STR9##
D-myo-inositol-1.2.6-triphosphate alternatively D-1.2.6-IP.sub.3
##STR10## D-myo-inositol-1.2.5-triphosphate alternatively
D-1.2.5-IP.sub.3 ##STR11## myo-inositol-1.2.3-triphosphate
alternatively 1.2.3-IP.sub.3 P = OPO.sub.3 H.sub.2 ##STR12##
L-myo-inositol-1.3.4-triphosphate alternatively L-1.3.4-IP.sub.3
##STR13## L-myo-inositol-1.2-diphosphate alternatively
L-1.2-IP.sub.2 ##STR14## D-myo-inositol-1.2.5.6-tetra- phosphate or
D-1.2.5.6-IP.sub.4 ##STR15## L-myo-inositol-1.2.3.4.5-penta
phosphate or L-1.2.3.4.5-IP.sub.5 P = OPO.sub.3 H.sub.2
______________________________________
These IP.sub.3 compounds are described and prepared in applicant's
simultaneously filed patent application Ser. No. 788,829 with the
title "Inositoltriphosphate".
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